Project Summary The high mortality rate of pancreatic ductal adenocarcinoma (PDAC) is primarily attributed to its aggressive nature for which the underlying biology remains elusive. Exosome microRNA (miRNA)-mediated intercellular communication is a critical signaling event in the tumor microenvironment that drives PDAC progression. While recent studies have shown that cancer exosomes selectively encapsulate certain miRNA species and deliver them to surrounding cells, very little is known about how exosome miRNA signaling is initiated in cancer cells and processed in the tumor microenvironment to promote tumor progression. In our preliminary studies we have observed that a group of miRNAs is selectively encapsulated and highly enriched in exosomes derived from PDAC cells, with miR-1246 being the highest enriched. Bioinformatics analysis revealed three miRNA motifs that are present in the miR-1246 sequence and commonly shared by the highly enriched exosome miRNAs. Further, using a biotin-labeled miR-1246 probe and proteomic analysis, we have identified SRSF1, TIA1, and EIF3B as the most abundant cellular proteins specifically associated with miR-1246. Based on these observations we hypothesize that RBPs interact with miRNA sequence motifs to control the selective encapsulation and enrichment of exosome miRNAs in PDAC cells, thereby initiating exosome miRNA signaling that promotes PDAC progression. We propose three specific aims to test this hypothesis. Specific Aim 1, To identify the miRNA sequence motifs that mediate exosome miRNA encapsulation in PDAC cells; Specific Aim 2, to determine whether SRSF1, TIA1, and EIF3B regulate selective exosome miRNA encapsulation in PDAC cells; Specific Aim 3, to examine whether exosome miR-1246 promotes PDAC progression in vivo. Upon completion of the proposed studies, we anticipate that we will have identified RBPs and miRNA motifs that mediate selective exosome miRNA encapsulation in PDAC cells, and determined the role of exosome miR- 1246 in promoting PDAC progression. A better understanding of how exosome miRNA signaling is initiated and processed will help establish new therapeutic strategies against PDAC.